This present invention relates to a method for activating a peroxidase enzyme system in situ.
A number of naturally occurring antimicrobial systems rely upon the ability of certain oxidizing agents to disrupt metabolic processes of bacteria, fungi and viruses. Examples of such oxidizing agents include hypothiocyanite (OSCNxe2x80x94/HOSCN), hypochlorite (OClxe2x80x94 HOCl), and hypoiodite (OIxe2x80x94 HOI). These agents are known to inhibit glycolysis, penetrate prokaryotic cell walls, and generally disrupt a wide variety of processes crucial to the survival of lower organisms at concentrations greater than or equal to about 100 micromoles per liter. The oxidizing agents are formed from the detoxification of hydrogen peroxide by mammalian peroxidase systems, such as those found in saliva, cervical fluid, lachrymal fluid, and leukocytes. Examples of such peroxidase system enzymes are myeloperoxidase, lactoperoxidase, and salivary peroxidase.
Attempts to exploit these natural antimicrobial systems have been directed to both the oral care field and the gastrointestinal tract. U.S. Pat. No. 4,150,113 and U.S. Pat. No. 4,178,362 (Hoogendorn, et al.) describe dentifrice compositions containing glucose oxidase that react with plaque and salivary glucose to produce low levels of hydrogen peroxide. Hydrogen peroxide production by such systems is, however, highly irregular due to the non-uniform distribution and unpredictable availability of substrate, namely glucose, in the oral cavity.
U.S. Pat. No. 4,269,822, U.S. Pat. No. 4,564,519 and U.S. Pat. No. 4,578,265 (Pellico, et al.) further describe dentifrice compositions containing an oxidoreductase enzyme and its specific substrate in an aqueous solution for the purpose of producing hydrogen peroxide or other antimicrobial oxidizing compounds such as hypothiocyanite ion. A more predictable amount of hydrogen peroxide (and subsequently hypothiocyanite ions) is produced by the compositions of Pellico et al., compared with those of the Hoogendorn references. The differences between the two compositions reflect the availability of glucose in the oral cavity as substrate for glucose oxidase.
There are, however, a number of disadvantages associated with the compositions of Pellico et al. These include: the limited rate of enzymatically-produced hydrogen peroxide that in turn produces the hypothiocyanite ion. The short duration of oral contact time, namely during toothbrushing, means that insufficient amounts of hypothiocyanite is available to effectively eliminate microbes in the oral cavity. In addition, the references utilize glucose oxidase as the oxidoreductase enzyme that in turn relies upon the availability of a sufficient concentration of glucose in solution to produce hydrogen peroxide. However, the glucose itself is a microbial substrate and is potentially cariogenic when present in an oral care product.
U.S. Pat. No. 4,564,519 describes a chewable dentifrice, such as a chewing gum or lozenge, which contains a dual enzyme system for producing hypothiocyanite ions upon being chewed or otherwise activated by the moisture in saliva. Such compositions suffer from similar drawbacks to those mentioned immediately above namely a slow rate of enzymatically-produced hydrogen peroxidase as well as a reliance on a cariogenic compound.
Other solid or chewable compositions capable of producing hydrogen peroxide or other oxidizing agents upon activation with moisture are taught in U.S. Pat. No. 4,320,116, U.S. Pat. No. 4,726,948, and U.S. Pat. No. 4,929,466. These compositions are foodstuffs intended for consumption by livestock in order to limit the growth of harmful bacterial within the animal""s gastrointestinal tract. These references describe the use of various enzymatic and non-enzymatic sources for hydrogen peroxide, where the enzymatic sources are glucose oxidase/glucose and the non-enzymatic sources are sodium perborate, sodium percarbonate, and calcium peroxide. However, it is known that sodium percarbonate and potassium percarbonate have extremely alkaline pH and are thus of little use in activating the peroxidase enzymes until exposed to the acidic environment within the gastrointestinal tract. Thus, the foodstuff compositions described in the reference cannot be used as a therapeutic or otherwise peroxidase-activating effect in the oral cavity.
It would thus be advantageous to provide substantially non-cariogenic compositions capable of rapidly producing hydrogen peroxide in conditions that are suitable for peroxidase enzyme activation in the oral cavity.
It would also be advantageous to provide compositions capable of rapidly producing antimicrobial hypohalite ions within the limited contact time available in most oral hygiene procedures.
It would also be advantageous to provide compositions capable of rapidly producing antimicrobial hypohalite ions upon contact with saliva within the limited contact time available in most oral hygiene procedures.
This invention satisfies the above needs. A novel oral care composition is provided.
A preferred embodiment of the invention is a non-enzymatic, water-soluble hydrogen peroxide precursor, capable of rapidly releasing an effective amount of hydrogen peroxide for activating the peroxidase system in the oral cavity, upon contact with an aqueous solution; and a pH adjusting agent capable of producing a selected pH in the aqueous solution for facilitating the rapid release of the hydrogen peroxide from the hydrogen peroxide precursor and the activation of the peroxidase enzyme in the oral cavity.
In a further embodiment of the invention, a process is provided for manufacturing an oral care product, comprising the steps of obtaining an alkali metal percarbonate; dispersing the percarbonate in a non-hygroscopic material so as to encapsulate the percarbonate; obtaining particles of percarbonate encapsulated in the non-hygroscopic material; associating the percarbonate particles with a pH adjusting agent; and formulating the particles into an oral care product.
In a further embodiment of the invention, a method is provided for activating a peroxidase system in an oral cavity of an animal, including the steps of selecting a non-enzymatic water soluble hydrogen peroxide precursor capable of rapidly releasing an effective amount of hydrogen peroxide for activating the peroxidase system in the oral cavity upon contact with an aqueous solution; mixing the precursor with a pH adjusting agent capable of producing a selected pH in the aqueous solution for facilitating the rapid release of the hydrogen peroxide from the hydrogen peroxide precursor and the activation of the peroxidase enzyme in the oral cavity; and administering to the oral cavity the precursor and pH-adjusting agent in a suitable formulation.
The present invention relates to oral care compositions which upon contact with an aqueous solution, are capable of rapidly activating a peroxidase enzyme so as to release hydrogen peroxide.
The concentrations of the components of the oral composition are given in molar units which denote the concentration of the component in the aqueous contact solution.
The limiting factor in all of the mammalian antimicrobial peroxidase systems is the availability of the substrate, namely hydrogen peroxide. Furthermore, the pH of the aqueous environment determines not only the effective release of hydrogen peroxide from the precursor but also the activity of the peroxidase system and the efficacy of the resulting oxidizing agents in penetrating the cell walls of microorganisms.
It is known, for example, that the non-ionized species of hypohalite ions more readily penetrates the cell walls of microorganisms then does the ionized species thereby having increased efficiency in inhibiting the metabolism of the microorganisms. The distribution of ionized versus non-ionized species (for instance HOCl, or hypochlorous acid, versus OClxe2x80x94, or hypochlorite ion) is highly pH dependent.
The pH activity profiles of the peroxidase enzymes lactoperoxidase, salivary peroxidase, and myeloperoxidase is maximum between pH 5 and 6, but drop off sharply below pH 4.0 and above pH 7.5. Thus, in order to maintain peroxidatic function, it is here concluded that the pH of the medium surrounding the peroxidase enzymes must be within the range of about pH 4.0 to about pH 7.5. This pH range also favors the anti-microbial, non-ionized hypohalite species which prevail at lower pH levels.
Consequently, the compositions of the invention include a non-enzymatic water-soluble hydrogen peroxide precursor and a water-soluble pH adjusting component capable of providing a pH to an aqueous contact solution of between about 4.0 and 7.9. The aqueous contact solution may commonly be saliva, but may also include an aqueous solution that is mixed with the precursor and pH adjusting agent prior to contact with the oral cavity.
The non-enzymatic water-soluble precursor may be selected from the group of stable persalts including, but not limited to, alkali metal percarbonates, for example, sodium and potassium percarbonate, alkali metal perborates, alkali metal peroxides, and hydrogen peroxide complexes such as carbamide peroxide. Preferred non-enzymatic hydrogen peroxide precursors are sodium percarbonate and carbamide peroxide due to their solubility characteristics and relatively benign toxicity in limited concentrations. The most preferred non-enzymatic hydrogen peroxide precursor is sodium percarbonate.
Sodium percarbonate is a relatively stable complex containing 2 moles of sodium carbonate complexed with 3 moles of hydrogen peroxide (27% hydrogen peroxide by weight). It is highly water soluble (120 grams per liter at 20xc2x0 C.) and produces a pH upon dissolution of between 10 and 11 (for a 1% solution). Thus, although sodium percarbonate possesses the desirable hydrogen peroxide-releasing properties for the practice of the present invention, alone they are of little utility for the activation of a peroxidase enzyme due to their high in-solution pH properties. Accordingly, a pH adjusting agent has been utilized to normalize the pH to a range of 4.0-7.9.
Carbamide peroxide is a 1 to 1 molar complex between urea and hydrogen peroxide (35% hydrogen peroxide by weight) with a molecular weight of 94.07. It is usually manufactured in the form of crystals which are highly soluble in water (800 grams per liter of water at 20xc2x0 C. to yield a saturated solution of 44.4% carbamide peroxide, equivalent to a hydrogen peroxide concentration of 15.5%). However, when carbamide peroxide is solubilized in water, a pH of approximately 3.40 (for a saturated solution) to approximately 4.05 (for a 1% solution) is obtained. This pH is slightly below the desirable range, according to the invention, for activating a peroxidase enzyme in the aqueous contact solution absent a pH adjusting agent.
The pH adjusting agent of the present invention may include any toxicologically acceptable and preferably water-soluble ingredient which is capable of producing an aqueous contact solution pH of between about 4.0 and about 7.5. Most preferably, the pH adjusting agent will provide a pH of between 5.0 and 6.0. Such pH adjusting agents include a wide variety of common buffers, acidulants, and/or alkalizers which are well known to those skilled in the art. Examples include organic acids and their alkali metal salts, such as citric acid, malic acid, butyric acid, gluconic acid, adipic acid, glutaric acid, and malonic acid; amines such as triethanolamine and tris(hydroxyaminomethane); alkali metal hydroxides, such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide; and combinations thereof. Preferably, these pH adjusting agents are free of water of hydration in order to achieve long-term stability in the presence of the hydrogen peroxide precursor. The optimum concentration of the pH-adjusting agent is the lowest level necessary to achieve the desired pH adjustment of the aqueous contact solution to between 4.0 and 7.5. This concentration of the pH adjusting agent is in the range of about 0.01% by weight of the composition to about 1% by weight of the composition. However, higher and lower amounts may have utility in circumstances where the buffering capacity of the surrounding medium is either very strong or very weak, respectively.
The oral care composition described above generates hydrogen peroxide to be used as substrate for the peroxidase system in a manner that permits the concentration of hydrogen peroxide generated overall to be reduced while increasing the rate of its production. This provides a safe and effective antimicrobial composition. While the prior art describes antimicrobial activity associated with oral care products containing or generating hydrogen peroxide, the present invention provides for the first time the particular advantages of both limiting the concentration of hydrogen peroxide to a level which is suitable for the activation of a peroxidase enzyme (less than about 10 millimoles per liter in situ) and providing or limiting the pH range of the in situ composition/saliva fluids to that which is also most advantageous for activation of a peroxidase enzyme.
The antimicrobial activity of the oral compositions depend on the presence of an oxygen acceptor. The preferred oxygen acceptor for oral care applications (i.e., in the presence of salivary peroxidase or lactoperoxidase) is the thiocyanate ion, which can be provided to the composition through the inclusion of non-toxic levels of a thiocyanate salt, such as potassium or sodium thiocyanate. In general, the level of thiocyanate salt included in said compositions will be from about 1.0 millimolar to about 10.0 millimolar (again, as above, based upon the concentration achieved in the aqueous contact solution). However, the composition of the invention may be further enhanced by incorporating halide ions in the aqueous contact solution. One or more oxygen-accepting halide or pseudohalide ions, including any of chloride, iodide, bromide, and thiocyanate and combinations thereof, may be incorporated in the aqueous contact solution. These ions may already be present in solution (such as thiocyanate ion, which is present in saliva), or alternatively they may be provided as auxiliary components in the inventive compositions.
In general, oral care product use results in a dilution, of composition components on the order of 1 part composition to from about 1 part aqueous contact solution to about 5 parts aqueous contact solution (from about 1 to 1 to about 1 to 5). It is desirable that the non-enzymatic hydrogen peroxide precursors should be present at a level sufficient to release a minimum amount of hydrogen peroxide of approximately 100 micromoles per liter. A preferred range of hydrogen peroxide released is in the range of from about 500 micromoles per liter to about 2,000 micromoles per liter. The determination of the amounts of hydrogen peroxide that are released from a given composition in vitro (for instance, under controlled conditions in contact with distilled water as a diluent) are relatively simple to predict. However, the determination of amounts of hydrogen peroxide released in vivo indicates that in vivo levels are well below those predicted in vitro. This difference may result from the destruction of hydrogen peroxide by salivary catalase, interaction of hydrogen peroxide with various organic matter and non-enzymatic reducing agents in saliva, and the destructive effect of dissolved metal ions in saliva. Thus, while an upper limit of 2.0 millimoles of hydrogen peroxide per liter is predictive of an in vitro effectiveness, concentrations as high as 30 millimoles per liter may be desirable to produce much lower observed in vivo concentrations of hydrogen peroxide.
It should also be noted that an accumulation of high concentrations of hydrogen peroxide (greater than about 0.1 percent or about 29 millimoles per liter) ate not desirable due to the evidence that hydrogen peroxide is mutagenic and can cause cellular DNA damage at elevated concentrations. The prior art describes broad concentration ranges of hydrogen peroxide as halving utility in oral hygiene and tooth whitening applications, but makes little reference to the potential harmful effects of hydrogen peroxide at concentrations, for instance, in the range of 1.5 to 3.0 percent by weight (441 to 882 millimolar).
Although the peroxidase enzyme may typically be present in the aqueous contact solution (such as salivary peroxidase, which is resent in saliva), additional peroxidase enzyme, preferably lactoperoxidase, may be included in the inventive compositions in a range of from about 10 ABTS (2,2xe2x80x2-Azinobis(3-ethylbenzthiazoline sulfonic acid) units per gram of composition to about 1,000 ABTS units per gram of composition [under the assay conditions described in Pruitt, et al., Analytical Biochemistry 191, pp. 278-286 (1990)].
In a preferred embodiment of the invention, compositions may be non-aqueous, dry, or otherwise substantially water-free mixtures, which can be applied or deposited on or within an orally acceptable carrier, such as a chewing gum, dental floss, anhydrous dentifrice, or animal chew. These compositions, once dissolved in the aqueous solution at a selected pH, are capable of producing, in the absence of additional enzyme preparations, hydrogen peroxide that results in the rapid activation of an antimicrobial peroxidase enzyme system in vivo.
The hydrogen peroxide precursors of the invention, such as the alkali metal percarbonates, may be processed with little or no loss of activity from moisture pick-up if, prior to being deposited onto or into an orally acceptable carrier, the alkali metal percarbonate is first dispersed in a non-hygroscopic fluid or solid in order to coat or encapsulate each particle of the percarbonate prior to being entered into a manufacturing process. Alkali metal percarbonates processed in this manner show very little degradation during processing cycles due to moisture absorption and/or temperature exposure. Although the concept of coating moisture-sensitive materials with water-insoluble or non-hygroscopic outer layers is not new to the art, the inventive aspect of the present invention stems from the requirement that said coating must necessarily consist of a moisture-resistant fluid which has mobility and will form a fluid interface in contact with bulk moisture. Only through the formation of a thin interfacial layer in contact with bulk moisture will the intimately admixed alkali metal percarbonate and non-hygroscopic fluid release the alkali metal percarbonate (as hydrogen peroxide and an alkali metal carbonate) into the neighboring aqueous phase.
Preferred non-hygroscopic fluids and solids are non-solvents for alkali metal percarbonates. These fluids are water-insoluble, yet low enough in viscosity to be readily dispersed into a thin film or interface in the presence of bulk moisture. Such non-hygroscopic liquids include, but are not limited to, mineral oils, vegetable oils, fatty esters, silicone fluids, fluorinated hydrocarbons, and fluorosilicones. Preferred non-hygroscopic solids are also water insoluble and must be capable of being melt processed at a temperature suitable for maintaining the stability of the alkali metal percarbonate. Suitable non-hygroscopic solids include, but are not limited to, waxy solids such as mineral oils, vegetable oils, fatty esters, silicone fluids, fluorinated hydrocarbons, fluorosilicones, stearic acid, glyceryl monostearate, paraffin wax, microcrystalline wax, and fatty alcohols. An example of an applicable melt process includes batch melt processing, whereby the non-hygroscopic solid carrier is simply melted in bulk, whereupon the alkali metal percarbonate is added and dispersed. Another example of an applicable melt process is the fluid-bed processing technique described in U.S. Pat. No. 4,421,669, whereby the alkali metal percarbonate (in the form of a powdered or granular particle) is floated on an airstream and subsequently sprayed with melted droplets of the non-hygroscopic solid carrier.
Both non-hygroscopic fluid and solid carriers may also include the pH-adjusting agents of the present invention, so as to simplify the application of both inventive components (i.e., the hydrogen peroxide precursor and the pH-adjusting agent) onto or into the oral care product delivery system. Alternatively, the hydrogen peroxide precursor and the pH-adjusting agent may be dispersed separately within two different non-hygroscopic carriers and subsequently applied onto or into the oral care product delivery system in a stepwise fashion.
The choice of non-hygroscopic fluid or solid carrier for the alkali metal percarbonate is dependent upon the final oral care product delivery system contemplated. Where the formulation is an animal chew, such as a rawhide animal chew, the percarbonate may be dispersed in a non-hygroscopic fluid carrier before being deposited on the surface of the chew. Where the formulation is a chewing gum, the alkali metal percarbonate may be dispersed in either a non-hygroscopic liquid or solid, to be subsequently added batchwise to the gum base and thence kneaded to achieve homogeneity. Alternatively, the finished chewing gum may be coated with a layer of either a liquid or solid dispersion of alkali metal percarbonate.